STUDIES ON LAPAROSCOPIC INTRAUTERINE INSEMINATION OF BARKI EWES (USING DIFFERENT INSEMINATION DOSES (AS COMPARED WITH CERVICAL INSEMINATION

STUDIES ON LAPAROSCOPIC INTRAUTERINE INSEMINATION OF BARKI EWES (USING DIFFERENT INSEMINATION DOSES (AS COMPARED WITH CERVICAL INSEMINATION

EL-BADRY, D.A.^*; AML Z. LEIL ^** and MONA H. SHAKER ^*

^*Artificial Insemination and Embryo TransferDept. Animal Reproduction Research Institute, Agriculture Research Center, Giza, Egypt

^**Dept. of Field Investigations, Animal Reproduction Research Institute, Agriculture Research Center, Giza, Egypt

Email: diyabadry@hotmail.com

INTRODUCTION

Artificial insemination has become an important adjunct for breeding certain domestic species because of its great advantage for exploiting the genetic potential of superior sires. It has been known for quite long time that cervical insemination in sheep though commercially widely used, does not render satisfactory fertilization rate (Amiridis and Cseh, 2012).The cervical canal of the ewe has a convoluted and tortuous structure, reflecting the presence of 5–7 annular folds or cervical rings (Kershaw et al., 2005; Kaabi et al., 2006) that prevent trans-cervical intrauterine insemination (Kaabi et al., 2006).

Laparoscopic approach for intrauterine semen deposition is an easy technique, however, giving acceptable fertilization rates in estrous synchronized ewes (Lymberopoulos et al., 2001; Naqvi et al., 2001; Evans et al., 2002; Hiwasa et al., 2009).The advantage of laparoscopic insemination is that the semen is deposited closer to the site of fertilization. Deep uterine insemination has been shown to be advantageous in several domestic species, such as sheep (Salamon and Maxwell, 1995; Wulster-Radcliffe et al., 2004), goats (Ritar and Salamon, 1983; Moore et al., 1988), cattle (Lopez-Gatius, 2000; Verberckmoes et al., 2004), horses (Morris and Allen, 2002), and pigs (Martinez et al., 2002; Rath, 2002; Watson and Behan, 2003), especially when sperm numbers are limited or sperm quality is suboptimal.

The site of insemination of ram semen has a major effect on pregnancy rate in sheep, with greater rates achieved following laparoscopic AI than following either transcervical (Wulster-Radcliffe et al., 2004) or cervical insemination (Fair et al., 2005).Using laparoscopy, the “cervical barrier” problem has been overcome and satisfactory fertility rate has been achieved by significant reduction in the number of spermatozoa per insemination (from 200-300 to 1-10 millions, or less for sex-sorted semen, Salamon and Maxwell, 2000).

The major objectives of this study were; to compare the pregnancy and lambing rates of ewes inseminated either cervically or by intrauterine laparoscopic insemination and to determine the minimal sperm dose per intrauterine insemination in order to maximize the genetic diffusion of males, without decreasing AI success.

MATERIALS and METHODS

Animals and treatment:

Thirty six mature, clinically healthy Barki sheep (3 rams and 33 ewes), aged 2.0 -2.5 years were assigned to the study. Animals were kept in the experimental farm of the Animal Reproduction Research Institute (ARRI), and they were divided into four groups, the first three groups (8 ewes in each) were inseminated laparoscopically with either 10 x 10⁷ (group 1), 20 x 10⁷ (group 2) or 40 x 10⁷ sperm/dose(group 3). The last group (group 4 containing 9 ewes) was trans-cervically inseminated with 80 x 10⁷ sperm/dose(Gordon, 1997). 

Collection of semen:

Semen was collected from trained rams with an artificial vagina that was adjusted to a proper condition, diluted at 30°C with Tris-based diluent to provide a sperm concentration of 80 x 10⁷/ml, cooled slowly and kept at 16°C for up to 6 h (Langford et al., 1979). Only rams with at least 70% motile spermatozoa and good progressive motility were used. Just before insemination, dilutions were made at 16°C using Tris-based diluent to provide concentrations of 80, 40, 20 and 10 x 10⁷/ml.

Synchronization of estrus:

The estrous cycles were synchronized using CIDR’s (EAZI-BREED, CIDR®, New Zealand), inserted for a period of 14 days (Sirjani et al., 2012). eCG (250 IU; Folligon; Germany) intramuscularly injected to ewes at the time of CIDR removal. The laparoscopic and cervical inseminations were performed on the 58^th hour after removal of CIDR (Bonev et al., 2005).

Laparoscopic artificial insemination:

Laparoscopic procedures were done using Wolf Laparoscope (Wolf Co., Germany) of 5 mm diameter, 33 cm length and 0^o scope viewing angle.  Automatic insufflator was used to deliver the CO₂ intraperitoneally (pressure 10 mmHg). Laparoscopic insemination, in details was described by Toni et al. (2012). Briefly, ewes were fasted and restricted access to water at least for 16 hours before laparoscopy, and injected intravenously with xylazine hydrochloride (Xylaject 2%, Adwia) at a dose rate of 0.05mg/kg b.w. Local anesthetic (Lidocaine 2%) was injected 10 minutes before the procedure was performed. The ewe was then placed in a laparoscopy cradle. The abdominal region was surgically prepared by shearing the wool and disinfecting the skin. Using the cradle, the ewe was positioned in a supine head-down (Trendelenburg) position to an approximate angle of 30^o (Fig 1). A scalpel blade was used to make a small skin incision in order to facilitate trocar penetration (Fig 2). The trocars and cannulae for introducing laparoscope and insemination pipette were inserted 7-10 cm ventral to the udder and 5-10 cm on each side of the midline (linea alba). The 5-mm Verus needle that was connected with the CO₂ is first introduced and the abdomen was slightly inflated to reduce the chance of injury to organs. Insertions of the first trocar and cannula should be well controlled and the sharp trocar was withdrawn as soon as the abdominal wall has been penetrated. The blunt cannula was pushed well into the abdomen, while the second trocar and cannula were inserted after inflation with CO₂. Endoscope and AI instrument went through the cannulae (Fig 3) and the uterus was located and fixed using the grasping forceps just ventral to the urinary bladder. Semen was deposited in the lumen of each uterine horn approximately halfway between the uterine bifurcation and the utero-tubal junction (Fig. 4). Instruments were withdrawn and putted into disinfectants between each animal.  An antibiotic spray was applied to the wounds before it was sutured. The sutures were removed 7 days later.

Cervical artificial insemination:

The cervix was located, via a speculum fitted with a light source. The cervix of the ewe is convoluted in structure and does not dilate during oestrus. As a result it was generally only possible to deposit the semen (80 x 10⁷ sperm) in the first fold of the cervix.

Pregnancy diagnosis:

Pregnancy rate (pregnant ewes/treated ewes ratio) was determined 35 days after AI by trans-abdominal ultrasonography examination using an Exagyne ECM, France) machine provided with a 6.5 MHz linear probe. Pregnancy loss in ewes was calculated as the number of ewes pregnant at Day 35 – the number of lambed ewes / number of ewes pregnant at Day 35.

Statistical analysis:

The recorded data among all treatment groups was analyzed by Chi square analysis. The level of significance was observed at 5% (Snedecor and Cochran, 1989).

RESULTS

Regarding the results of laparoscopic intrauterine insemination (table 1), the insemination dose of 20 x 10⁷ spermatozoa (group 2) and 40 x 10⁷ spermatozoa (group 3) resulted in the same pregnancy rate at 35 days post-insemination (75.00% for each). However, insemination of 20 x 10⁷ spermatozoa resulted in significantly (P ≤ 0.05) higher lambing rate when compared with using 10 x 10⁷ and 40 x 10⁷ spermatozoa (75.00% vs. 37.50 and 62.50%, respectively). On the other hand, ewes of group 1 had the significantly higher rate of pregnancy loss (40.00%) when compared with rates of groups 2 and 3 (0.00% and 16.67%, respectively). 

The results presented in table (1) revealed that, laparoscopic intrauterine insemination using optimum insemination dose (20 x 10⁷ spermatozoa, group 2) was superior to cervical insemination using 80 x 10⁷ spermatozoa (group 4) in terms of pregnancy rate (75.00% vs. 55.56%, respectively) and lambing rate (75.00% vs. 55.56%, respectively).

Table 1: Pregnancy and lambing rates of ewes after cervical and laparoscopic intrauterine insemination using different insemination doses.

Values with different superscripts in the same raw differs significantly at least at P < 0.05

DISCUSSION

In the current study, the lambing rate after laparoscopic insemination using 20 x 10⁷ spermatozoa (group 2) was significantly (P ≤ 0.05) higher than that after cervical insemination using 80 x 10⁷ spermatozoa (75.00% vs. 55.56%, respectively). Similarly, Rojero et al. (2009) concluded that middle fertility rate of 43.7% resulting from cervical insemination in ewes can be considered as acceptable, but it is no possible to obtain similar fertility rate (75.00%) as with laparoscopic intrauterine insemination. Artificial insemination techniques have been considered in many previous studies. According to several authors (Armstrong and Evans, 1984; Rodriguez et al., 1988; Correa et al., 1994; Byrne     et al., 2000; Romano, 2013),laparoscopic insemination ensures significantly higher parturition rates than trans-cervical insemination, despite the fact that relatively lower numbers of spermatozoa are used. This difference in fertility can be explained by the fact that the sheep cervix has a very high structural complexity, preventing deep cervical insemination (Halbert et al., 1990; Kaabi, 2002). Laparoscopic insemination allows this barrier to be bypassed, improving fertility even with lower quality spermatozoa (Salamon and Maxwell, 2000; Naqvi     et al., 2001).  

The lambing rate of 75.00% achieved after laparoscopic intrauterine insemination in present study coincided with the same rates (75.00%) reported by McKelvey et al. (1985) and Rojero et al. (2009),and higher than lambing rates of 48.00%, 71.10%, 43.90%, 72.70%, 60.00%, 71.00% and 71.40% reported by Windsor et al. (1994), Hill et al. (1998), McKusick et al. (2000); Paulenz et al. (2005), Toni et al., 2012, Al-Wataar (2009) and Alfaris et al. (2012), respectively. Sayre and Lewis (1997)reported a higher lambing rate (92.50%) after intrauterine insemination. Concerning the cervical insemination, the lambing rate of 55.56% achieved after cervical insemination in this study was similar to that (57.00%) reported by Ghalsasi and Nimbkar (1996),and lower than lambing rates of 68.60%, 78.00%, 69.00%, 67.00%, 65.75% and 60.00% reported by Lightfoot and Salamon (1970), Langford et al. (1979), Tervit et al. (1984), McKelvey et al. (1985), Donovan et al. (2000), Nour et al. (2010), respectively,and higher than lambing rates of 43.70% and 50.00% reported by Rojero et al. (2009) and Al-Wataar (2009), respectively.The fertility rates following cervical and laparoscopic insemination all vary with the insemination technique used as well as with the farm, age, male, number of insemination per ewe, lambing-insemination interval, technician, flock and management conditions (Anel et al., 2005; Paulenz et al., 2005).

In sheep, the numbers of spermatozoa used by intrauterine insemination were reported to be 80 million (Windsor et al., 1994), 150 million (Halbert  et al., 1990; Buckrell et al., 1994), 200 million, (Lawrence, 1985; Husein et al., 1998a) and 400 million (Smith et al., 1995; Husein et al., 1998b). In general, the numbers of spermatozoa used for intrauterine insemination are higher than the recommended numbers used for trans-cervical artificial insemination (Maxwell and Hewitt, 1986; Ritar, 1993; Romano, 2013).In the current study, a low intrauterine insemination dose (10×10⁷) resulted in decreased lambing rate (37.50%), whereas a higher insemination dose (20×10⁷) increased lambing rate (75.00%) and this result was in agreement with other researches (Maxwell and Salamon, 1993; Martin and Watson, 1976; Emsen et al., 2011). Higher dose of spermatozoa (40×10⁷) was not recorded with significant increase in lambing rate (62.50%). This result came in accordance with the findings of Emsen et al. (2011). Thus, it can be recommended that the minimum necessary for laparoscopic artificial insemination in Barki ewes is 20×10⁷ motile spermatozoa. Similarly, Milczewski et al. (2000) recommended that higher pregnancy rates (69.56%) could be obtained with at least 25 x 10⁷ spermatozoa per dose in intrauterine inseminations of ewes. On the other hand, Leethongdee (2010) recommended a minimum number of 40 x 10⁶ spermatozoa per laparoscopic insemination.  Also, Evans and Maxwell (1987) recommend a minimum dose of only 20 x 10⁶ motile sperm while there are several reports of acceptable fertility (> 50%) using doses as low as 5 x 10⁶ (Eppleston et al., 1986)and 10 x 10⁶ (Salamon   et al., 1985) motile spermatozoa. Furthermore, acceptable levels of fertility were achieved after low-dose insemination using flow cytometrically sorted ram sperm at a dose of 1 x 10⁶ motile sperm per ewe (de Graaf et al., 2007).

In conclusion, as compared to cervical insemination, intrauterine insemination using the technique of laparoscopy is a relatively simple and convenient mean of achieving high lambing rates in Barki ewes. The recommended insemination dose for laparoscopic artificial insemination of Barki ewes was 20×10⁷ motile spermatozoa.

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